This is a 371 of PCT/EP97/01 013, filed Feb. 28, 1997.
The present invention relates to peptide immunogens, and is directed to inhibition of interactions which would normally cause the triggering of mast cells and basophils induced by cell-bound IgE linked to,an allergen resulting in the release of pharmacologically active mediators as well as the de novo synthesis of cytokines involved in the regulation of allergic and inflammatory reactions. It concerns immunogenic molecules including a moiety of a BSW 17 mimotope peptide and their use.
Allergic symptoms are brought about through the release of pharmacologically active mediators, notably histamine, leukotrienes and enzymes, from cells into surrounding tissue and vascular structures. These mediators are normally stored or synthesized de novo in special cells known as mast cells and basophil granulocytes. Mast cells are dispersed throughout animal tissue whilst basophils circulate within the vascular system. These cells synthesize and store mediators within the cell, unless a specialized sequence of events occurs to trigger its release.
The role of immunoglobulin E (IgE) antibodies in mediating allergic reactions is well known. IgE is a complex arrangement of polypeptide chains which, as in other immunoglobulins consists of two light and two heavy chains linked together by disulphide bonds in a xe2x80x9cYxe2x80x9d shaped configuration. Each light chain has two domains, one variable (VL) domain linked to a domain with a relatively invariant amino acid sequence termed a constant domain (CL). Heavy chains, by contrast, have one variable domain (VH) and in the case of IgE, four constant domains (CH1, CH2, CH3, CH4, also known as Cxcex51, Cxcex52, Cxcex53, Cxcex54). The two xe2x80x9carmsxe2x80x9d of the antibody are responsible for antigen binding, having regions where the polypeptide structure varies, and are termed Fabxe2x80x2 fragments or F(abxe2x80x2)2, which respresents two Fabxe2x80x2 arms linked together by disulphide bonds. The xe2x80x9ctailxe2x80x9d or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment. The Fc fragment contains interactive sites which enable the antibody to communicate with other immune system molecules or cells by binding to their Fc receptors.
Fc receptors are molecules which bind specifically to active molecular sites within immunoglobulin Fc regions. Fc receptors may exist as integral membrane proteins within a cell""s outer plasma membrane or may exist as free xe2x80x9csolublexe2x80x9d molecules which freely circulate in blood plasma or other body fluids. In the human system, high affinity binding of IgE to the receptor Fcxcex5RI is accomplished by a complex protein-protein interaction involving various parts of the third heavy chain constant region domain (Cxcex53) of IgE, and the membrane-proximal, immunoglobulin-like domain (xcex12) of the Fcxcex5RIxcex1 subunit.
Although residues within the Cxcex53 domain of the IgE heavy chain constant region, and regions belonging to the xcex12 domain of the Fcxcex5RIxcex1 receptor, have been identified which are important for binding, the detailed mechanism of the binding process are still obscure. Experimental evidence has been provided by fluorescence energy transfer measurements as well as X-ray and neutron scattering that human IgE adopts a bent structure which is speculated to contribute to the uniquely high affinity of IgE for Fcxcex5RI (Kdxcx9c10xe2x88x9210 M). Moreover, this bent structure is also postulated to be responsible for the equimolar complex between IgE and cell bound or soluble Fcxcex5RIxcex1, although the IgE molecule would provide identical epitopes on the two Cxcex53 domains for receptor binding. This monovalency is a functional necessity if receptor triggering in the absence of allergen is to be avoided (FIG. 1). Interactive sites, depending on their function, may already be exposed and therefore able to bind to cellular receptors. Alternatively, they may be hidden until the antibody binds to the antigen, whereupon the antibody may change in structure and subsequently expose other active sites which can then trigger a specific immune activity. Based on data obtained from circular dichroism spectra, a conformational rearrangement affecting Cxcex53 upon receptor binding has been proposed as an explanation for the 1:1 stoichiometry of the Fcxcex5/Fcxcex5RI complex on the cellular surface.
For allergic (immunological) release of histamine within the organism from mast cells and basophils, an IgE molecule must lock onto or attach itself with its Fc portion to the cellular Fc receptor site, thus securing the IgE molecule to the mast cell or basophil. The Fabxe2x80x2 portions of the cell-bound IgE molecules must be cross-linked by a particular compatible antigen (the allergen). When such an interaction occurs, the mast cell or basophil is automatically triggered to release histamine to the local environment, manifesting familiar allergic symptoms. Other biochemical events follow in a late phase reaction, resulting in de novo synthesis and release of cytokines and other mediators [Ravetch, J. V., and J. P. Kinet, Ann. Rev. Immunol. 9 (1991) 457-492].
Conventional approaches to allergy treatment have involved systemic therapy with anti-histamines or steroids or attempts to desensitize patients; these approaches are not adressed to the basic IgE-mast cell/basophil interaction. Other approaches have been concerned with the production of polypeptide chains capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound. Moreover, investigations have been carried out in order to define the nature of a putative xe2x80x9ceffectorxe2x80x9d site within the IgE Fc region, which was speculated to provide an immunological signal which triggers mast cells/basophils for mediator release.
Using recombinant IgE fragments as immunogens for the generation of a protective anti-IgE vaccine has also been tried and shown to be effective. The main argument against such a vaccine results from the fear that using large IgE fragments for immunization could initiate not only the production of inhibitory antibodies but also generate crosslinking and thereby anaphylactogenic antibodies in the patients (FIG. 2).
A strategy to overcome this problem would aim at the identification of the smallest IgE fragment possible, ideally consisting of the receptor binding site only, which is buried within the IgE/Fcxcex5RI complex after binding and therefore no longer accessible for crosslinking by the vaccine-generated immune response. Attempts to reconstruct such a complex molecular entity seem unlikely to be successful in view of the spatial distances of the various Cxcex53 regions involved in IgE/Fcxcex5RI interaction.
It has now been found that the problems intrinsically linked to the xe2x80x9cclassicalxe2x80x9d vaccine approach are overcome by using BSW17 mimotopes for active immunization, either as chemically synthesized peptides coupled to appropriate carriers, or as recombinant fusion constructs (e.g. with ovalbumin, IgG, etc.).
BSW17 is a monoclonal antibody which recognizes a conformational epitope on Fcxcex5 with at least part of it residing within Cxcex53. The hybridoma cell-line producing monoclonal antibody BSW17 has been deposited on Dec. 19,/1996 with ECACC Salisbury, Wilthuv, SP4 OJE, United Kingdom under the provisions of the Budapest Treaty on the deposit of microorganisms, under deposit number 96121916. This antibody displays an interesting profile of biological activities, as summarized in FIG. 3. BSW17 or BSW17-like antibodies circulating within the vascular system protect from allergic reactions by
a) inhibiting the triggering of mast cells and basophils through competitive inhibition of the IgE/IgERI interaction and
b) lowering serum IgE levels through downregulation of IgE synthesis at the B cell level.
BSW17 xe2x80x9cmimotopexe2x80x9d peptides have now been identified by random peptide phage display library screening, i.e. peptides which mimic at least part of the complex conformational epitope on the IgE molecule. Chemically synthesized mimotope peptides coupled to an immunogenic carrier protein can be used e.g. as vaccines for the specific generation of antibodies in an allergic host which inhibit mast cell/basophil triggering by blocking IgE/Fcxcex5RIxcex1 binding and/or IgE synthesis. As mimotopes of an anti-IgE antibody they induce an immune response which results in the production of BSW17-like antibodies in the host. Since BSW17 has been shown to be non-anaphylactogenic, inhibitory to IgE/Fcxcex5RI binding and IgE synthesis on B cells, these antibodies raised against the BSW17 mimotope-based vaccines have analogous protective properties. The immune response is very specific since, in contrast to the xe2x80x9cclassical vaccine, approachxe2x80x9d, no IgE-derived protein fragments are present which could generate crosslinking antibodies in the immunized patients (FIG. 4).
The invention thus concerns immunogenic molecules comprising
(a) at least one moiety of a BSW17 mimotope peptide and
(b) a moiety capable of eliciting an immune response against that peptide, hereinafter briefly named xe2x80x9cthe immunogens of the inventionxe2x80x9d.
Component (a) preferably consists of up to five, preferably one or two, especially one moiety of a BSW17 mimotope peptide. Component (b) preferably is a conventional immunogenic carrier as set out hereunder, especially BSA or KLH.
The BSW17 mimotope peptide in component (a) preferably is up to about 15 amino acids altogether, it is e.g. one of the sequences (A) to (Q) (Seq.id.no. 1 to no. 17) hereafter. However, it may appropriately include further components for hapten-carrier binding, e.g. to facilitate coupling to component (b) or further processing. Thus, when the BSW17 mimotope peptide is cyclic, the two ends can e.g. be held together by two additional cystein residues forming a disulfide bridge, or the ends can be chemically crosslinked, e.g. with lysine; or when the BSW17 mimotope peptide is linear, the carboxy terminal amino acid may conveniently be blocked by amidation, and/or the amino terminal amino acid may conveniently be blocked by acetylation. Further, the BSW17 mimotope peptide moieties in component (A), e.g. the preferred moieties (A) to (Q) hereafter, may be flanked in the immunogens of the invention by a few, preferably one or two, additional ancillary groups, such as acetyl, cysteine or lysine, and/or an additional coupling group, such as DC or BSS, e.g. as set out for the specific immunogens of the invention disclosed in Example 8 hereunder as conjugates (2) to (4), (6) to (11), (13) and (14).
The antibodies elicited by the immunogens of the invention, in contrast to the antibodies produced by hybridoma BSW17, will be endogenous and thus, in a patient, human; they may be used for prophylactic treatment.
They may be prepared by appropriately coupling components (a) and (b) as defined above.
The immunogens of the invention are e.g. in the form of a polymeric peptide or a recombinant fusion protein, whereby a monomeric component of the polymeric peptide, or one partner of the fusion protein, constitutes a moiety of a BSW17 mimotope peptide (a) and the remainder of the polymeric peptide or fusion protein constitutes the immune response-eliciting moiety (b).
They especially are in the form of a conjugate of at least one BSW17 mimotope peptide moiety (a) and an immunogenic carrier moiety (b).
Preferred BSW17 mimotope peptide moieties, i.e. component (a), of the immunogens of the invention essentially consist of or contain an amino acid sequence selected from
More preferred are (A), (D) and (G) above, especially (A) and (D).
The invention also concerns pharmaceutical compositions, especially vaccines, comprising immunogen molecules as defined above and an adjuvant.
It also concerns ligands, i.e. antibodies or fragments derived therefrom directed against BSW17 mimotope peptides used in xe2x80x9cpassive immunizationxe2x80x9d (see below), whereby the antibody or antibody fragments also recognize the natural epitope for BSW17 on human IgE; namely, it concerns ligands comprising an antibody domain specific for a moiety of a BSW17 mimotope peptide as defined above, whereby the antibody domain is reactive also with the sequence of amino acids on the heavy chain of IgE which comprises the natural epitope recognized by BSW17. Such ligands can be generated in mammals as polyclonal or monoclonal antibodies; they preferably are in the form of monoclonal antibodies, preferably in the form of an Fabxe2x80x2 fragment or an F(abxe2x80x2)2 fragment thereof.
It further concerns a process for the preparation of an immunogen of the invention, comprising covalently coupling
a) at least one moiety of a BSW17 mimotope peptide with
b) a moiety capable of eliciting an immune response against that peptide.
It also concerns immunogenic molecules as defined above, for use as a pharmaceutical, e.g. in the treatment of IgE-mediated diseases such as allergy and atopic dermatitis.
It further concerns the use of immunogenic molecules as defined above in the preparation of pharmaceutical compositions, especially vaccines, against IgE-mediated diseases, in particular allergy and atopic dermatitis.
It further concerns a method of prophylactic or curative immunization against IgE-mediated diseases such as allergies and atopic dermatitis comprising the administration of a therapeutically effective amount of immunogenic molecules as defined above to a patient in need of such treatment.
The immunogens of the invention, while being substantially incapable of mediating non-cytolytic histamine release, are capable of eliciting antibodies with strong serological cross-reactivity with the target amino acid sequences of the Fc region of IgE. They are thus useful in, or as, vaccines.
The initial dose of immunogen (e.g. from about 0.2 mg to about 5 mg, especially about 1 mg) is for example administered intramuscularly, followed by repeat (booster) doses of the same 14 to 28 days later. Doses will of course depend to some extent on the age, weight and general health of the patient. Immunization may be xe2x80x9cactivexe2x80x9d or xe2x80x9cpassivexe2x80x9d.
In xe2x80x9cactivexe2x80x9d immunization the patient receives immunogen of the invention and thereby an anti-hIgE response is actively induced by the patient""s immune system.
xe2x80x9cPassivexe2x80x9d immunization is achieved by administering anti-BSW17 mimotope antibodies, either polyclonal or monoclonal, to a patient suffering from IgE-mediated disease, preferably by injection.
Polyclonal anti-BSW17 mimotope antibodies can be prepared by administering immunogen of the invention, preferably using an adjuvant, to a non-human mammal and collecting the resultant antiserum. Improved titres can be obtained by repeated injections over a period of time. There is no particular limitation to the species of mammals which may be used for eliciting antibodies; it is generally preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, rats, cows, sheep, etc., can also be used. In the production of antibodies, a definite amount of immunogen of the invention is e.g. diluted with physiological saline solution to a suitable concentration and the resulting diluted solution is mixed with complete Freund""s adjuvant to prepare a suspension. The suspension is administered to mammals, e.g. intraperitoneally, e.g. to a rabbit, using from about 50 xcexcg to about 2500 xcexcg immunogen of the invention per administration. The suspension is preferably administered about every two weeks over a period of up to about 2-3 months, preferably about 1 month, to effect immunization. Antibody is recovered by collecting blood from the immunized animal after the passage of 1 to 2 weeks subsequently to the last administration, centrifuging the blood and isolating serum from the blood.
Monoclonal anti-BSW17 mimotope antibodies may e.g. be human or murine. Preferably, the patient will be treated with an Fabxe2x80x2 fragment preparation from murine monoclonal antibody or a chimeric human-mouse antibody (comprising human Fc region and mouse Fabxe2x80x2 region) so as to minimize any adverse reaction to the foreign animal immunoglobulin. Murine monoclonal antibodies may be prepared by the method of Kxc3x6hler and Milstein (Kxc3x6hler, G. and Milstein, C., Nature 256 [1975] 495), e.g. by fusion of spleen cells of hyperimmnunized mice with an appropriate mouse myeloma cell line. Numerous methods may be utilized to raise human monoclonal antibodies, including production by:
(1) Epstein-Barr virus (EBV)xe2x80x94transformed B-cells;
(2) cell line for B lymphocyte hybridization;
(3) human-murine hybridomas;
(4) human-human hybridomas;
(5 ) human x human-mouse heterohybridomas; and
(6) repertoire cloning (phage display).
Human x human-mouse heterohybridomas are the most preferred, and involve combining favourable characteristics of both human and murine parental cell types. Human-mouse heterohybridoma cell lines have been rendered suitable for B cell fusion (Teng, N. N. M. et al., Proc. Natl. Acad. Sci. USA 80 [1983] 7308).
When used for immunization, antibody can be introduced into the host most conveniently by intramuscular injection. Any conventional liquid or solid vehicle may be employed which is acceptable to the host and does not have adverse side effects on the host nor detrimental effects on the vaccine. Phosphate-buffered saline (PBS), at a physiological pH, e.g. about pH 6.8 to 7.2, preferably about pH 7.0, may be used as a vehicle, alone or with a suitable adjuvant, such as an aluminium hydroxide-based adjuvant. The concentration of immunogenic antigen may vary from about 50 xcexcg to about 500 xcexcg, preferably from about 200 xcexcg to about 300 xcexcg per injection, in a volume of solvent generally of from about 0.25 ml to about 1 ml, preferably about 0.5 ml. Multiple injections will be required after the initial injection and may be given e.g. at annual intervals.
As regards xe2x80x9cactivexe2x80x9d immunization, this is preferred for human use, but other mammalian species may be treated similarly, using analogous mimotopes corresponding to the IgE of these specie, as e.g. in the dog. The term xe2x80x9cimmunogenic carrierxe2x80x9d herein includes those materials which have the property of independently eliciting an immunogenic response in a host animal and which can be covalently coupled to polypeptide either directly via formation of peptide or ester bonds between free carboxyl, amino or hydroxyl groups in the polypeptide and corresponding groups on the immunogenic carrier material, or alternatively by bonding through a conventional bifunctional linking group, or as a fusion protein.
Examples of such carriers include: albumins, such as BSA; globulins; thyroglobulins; hemoglobins; hemocyanins (particularly Keyhole Limpet Hemocyanin [KLH]); proteins extracted from ascaris, e.g. ascaris extracts such as those described in J. Immun. 111 [1973] 260-268, J. Immun. 122 [1979] 302-308, J. Immun. 98 [1967] 893-900, a Am. J. Physiol. 199 [1960] 575-578 or purified products thereof; polylysine; polyglutamic acid; lysine-glutamic acid copolymers; copolymers containing lysine or ornithine; etc. Recently, vaccines have been produced using diphteria toxoid or tetanus toxoid as immunogenic carrier material (Lepow M. L. et al., J. of Infectious Diseases 150 [1984] 402-406; Coen Beuvery, E. et al., Infection and Immunity 40 [1983] 39-45) and these toxoid materials can also be used in the present invention. The purified protein derivative of tuberculin (PPD) is particularly preferred for utilization in the xe2x80x9cactivexe2x80x9d immunization scheme since (I) it does not induce a T-cell response itself (i.e. it is in effect a xe2x80x9cT-cell haptenxe2x80x9d), and yet behaves as a fully processed antigen and is recognized by T-cells as such; (2) it is known to be one of the most powerful hapten xe2x80x9ccarriersxe2x80x9d in the linked recognition mode; and (3) it can be used in humans without further testing.
As hapten-carrier binding agents, those conventionally employed in the preparation of antigens can be employed, e.g. those set out above, or in the Examples hereunder.
The process of the invention for covalently coupling component (a) to moiety (b) can be effected in known manner. Thus, for example, for direct covalent coupling it is preferred to utilize bis-N-succinimidyl derivatives, more preferably bis(sulfosuccinimidyl)suberate (BSS) as coupling agent. Glutaraldehyde or carbodiimide, more preferably dicyclohexyl-carbodiimide (DC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide may also be used for covalent coupling of peptide (a) to immunogenic carrier material (b).
The amounts of hapten and hapten-carrier binding agent [i.e. component (a)] and carrier [i.e. component (b)] can be readily ascertained in conventional manner. It is preferred that the carrier be employed in an amount of about 1 to about 6 times, preferably about 1 to about 5 times the weight of the hapten, and the hapten-carrier binding agent be employed in an amount of about 5 to about 10 times the molar equivalent of the hapten. After reaction, the carrier is bound to the hapten via the hapten-carrier binding agent to obtain the desired antigen composed of a peptide-carrier complex. The resultant immunogen of the invention can be readily isolated in conventional manner, e.g. by dialysis, gel filtration, fractionation precipitation, etc.
The preparation of the starting materials may be effected in conventional manner. Appropriate peptides for use as component (a) may e.g. be identified by screening of random peptide phage display libraries, and readily synthesized e.g. by conventional solid phase procedures, e.g., for cyclic peptides, by the solid phase procedure employing the well-known xe2x80x9cF-mocxe2x80x9d procedure, or may alternatively be identified using a peptidomimetic strategy by screening of randomly synthesized peptides.